Plasmid vector and a method for regulation of gene expression using the same

ABSTRACT

A plasmid vector capable of replicating in a Coryneform bacterial cell bearing a base sequence (a) functioning as an promoter in a Coryneform bacterium, a base sequence (b) functioning as an operator downstream from the base sequence (a), a base sequence (c) functioning as a site for ribosome binding in a Coryneform bacterial cell, a base sequence (d) functioning as a translation initiation codon, and a gene to be expressed which is directly ligated with the base sequence (d) and bearing a gene coding for a repressor protein capable of binding to the base sequence (d) functioning as an operator.

This is a continuation, of application Ser. No. 08/167,112 filed on Dec.16, 1993 now U.S. Pat. No. 5,426,050 which is a continuation of Ser. No.08/035,502 filed on Mar. 22, 1993 now abandoned, which is a continuationof Ser. No. 07/774,374 filed on Oct. 10, 1991 now abandoned, which is acontinuation of Ser. No. 07/339,876 filed on Apr. 18, 1989 nowabandoned, which is a continuation of Ser. No. 06/901,642 filed on Aug.29, 1986 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for controlling phenotypicgene expression of Coryneform bacteria.

2. Description of the Prior Art

Coryneform bacteria are microorganisms which are industrially important.Many Coryneform bacteria which produce large quantities of L-glutamicacid are known, including their mutants producing amino acids such aslysine, etc., and purine nucleotides such as inosinic acid, etc.

On the other hand, breeding and improvement of microorganisms forindustrial use utilizing recombinant DNA techniques have been recentlyattempted in, for example, Escherichia coli. With respect to Coryneformbacteria, some vectors are known as growing in these microorganisms as ahost and expressing chemical resistance as a marker (e.g., PublishedEuropean Patent Application No. 93611), but no method for artificiallyregulating inserted foreign genes has been discovered. Therefore, it hasbeen difficult to regulate gene expression artificially while expressingforeign genes using Coryneform bacteria as a host.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide aplasmid vector capable of regulating foreign gene expression inCoryneform bacterial cells.

It is a further object of this invention to provide a method forregulating the expression of foreign genes in Coryneform bacterialcells.

These and other objects of the present invention as will hereinafterbecome more readily apparent have been accomplished by providing aplasmid vector capable of replicating in a Coryneform bacterial cellhaving a base sequence (a) functioning as a promoter in the Coryneformbacterium, a base sequence (b) functioning as an operator downstreamfrom the base sequence (a), a base sequence (c) functioning as a sitefor ribosome binding in a Coryneform bacterial cell, a base sequence (d)functioning as a translation initiation site, and a gene to beexpressed, which is directly ligated with base sequence (d) and bears agene coding for a repressor protein capable of binding to the basesequence (b) functioning as an operator.

The invention further comprises a method for regulating gene expressionin Coryneform bacterial cells, which involves using a Coryneformbacterial cell having a plasmid vector comprising base sequences (a)-(d)and the gene to be expressed which is directly ligated with basesequence (d) and bearing a gene coding for a repressor protein.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention, and many of the attendantadvantages thereof will be readily obtained as the invention becomesbetter understood by reference to the following detailed descriptionwhen considered in connection with the accompanying drawings, wherein:

FIG. 1 is a drawing explaining the procedure for constructing apromoter-detecting vector pEB 001. The abbreviations have the followingmeanings:

P: Pst I

C: Cla I

H: Hind III

B: BamH I

Km^(r) : kanamycin-resistant gene

Cm^(s) : structural gene region of chloramphenicol-resistant gene

FIG. 2 is a drawing explaining the procedure for constructing apromoter-detecting vector pEB 003. The abbreviations have the followingmeanings:

P: Pst I

C: Cla I

H: Hind III

B: BamH I

S: Sal I

Km^(r) : kanamycin-resistant gene

Cm^(s) : structural gene region of chloramphenicol-resistant gene

FIG. 3 shows a structure of DNA fragment C used for the formation of apromoter-detecting vector pEB 003.

FIG. 4 is a drawing explaining the procedure for constructing a geneexpression-regulating vector pEC 701.

FIG. 5 is a drawing explaining the procedure for constructing a geneexpression-regulating vector pEC 702.

FIG. 6 is a drawing explaining the procedure for constructing a geneexpression-regulating vector pEC 801.

FIG. 7 is a drawing explaining the procedure for constructing a geneexpression-regulating vector pEC 901.

FIG. 8 shows the structure of trimethoprim-resistant vector pAJ 228.

FIG. 9 is a drawing explaining the procedure for constructing a geneexpression-regulating vector pEC 830.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Coryneform bacteria are aerobic, gram-positive rods, non-acid fast anddescribed in Bergey's Manual of Determinative Bacteriology, 8th edition,page 599 (1974). Examples of wild strains of Coryneform bacteria whichcan be utilized as host bacteria in the present invention include thefollowing:

Brevibacterium divaricatum ATCC 14020,

Brevibacterium saccharolyticum ATCC 14066,

Brevibacterium immariophilum ATCC 14068,

Brevibacterium lactofermentum ATCC 13869,

Brevibacterium roseum ATCC 13825,

Brevibacterium flavum ATCC 13826,

Brevibacterium thiogenitalis ATCC 19240,

Corynebacterium acetoacidophilum ATCC 13870,

Corynebacterium acetoglutamicum ATCC 15806,

Corynebacterium callunae ATCC 15991,

Corynebacterium glutamicum ATCC 13032, 13060,

Corynebacterium lilium ATCC 15990,

Corynebacterium melassecola ATCC 17965, and

Microbacterium ammoniaphilum ATCC 15354.

Coryneform bacteria include mutants which are derived from theseglutamic acid-producing bacteria or have lost glutamic acidproductivity, and mutants which produce amino acids such as lysine,arginine, etc., purine nucleosides such as inosine, etc., purinenucleotides such as inosine-5'-mono-phosphate, etc. and other products.Any plasmid that proliferates in Coryneform bacterial cells can be usedas the plasmid capable of replicating in Coryneform bacterial cells.Examples of wild strains of such a plasmid include the following:

(1) pAM 330: cf. Published Unexamined Japanese Patent Application67699/83

(2) pHM 1519: cf. Published Unexamined Japanese Patent Application77895/83

(3) pCG 1: cf. Published Unexamined Japanese Patent Application134500/82

(4) pCG 2: cf. Published Unexamined Japanese Patent Application 35197/83

(5) pCG 4: cf. Published Unexamined Japanese Patent Application183799/82.

As the plasmid capable of replicating in Coryneform bacterial cells, itis sufficient to have contained therein an initiation site forreplication of these wild plasmids. Therefore, there is no particularproblem even though the vector contains DNA's other than the initiationsite for replication.

Any sequence can be used as the base sequence (a) functioning as apromoter as long as it is a base sequence functioning as a promoter inCoryneform bacterial cells. Of course, the base sequence can be a basesequence of a promoter derived from Coryneform bacteria and basesequences of foreign promoters such as base sequences of variouspromoters derived from Escherichia coli (D. K. Hawley and W. McClure,Nucleic Acids Research 11, 2237-2255 (1983)), and others.

The base sequence (b) functioning as an operator can be any basesequence as long as the repressor protein can be bound thereto. Ofcourse, the base sequence (b) includes a base sequence of an operatorderived from Coryneform bacteria, base sequences of various operatorsderived from Escherichia coli, e.g., base sequences of foreign operatorssuch as a lac operator, a trp operator (Kenzo Nakamura,KAGAKU-NO-RYOIKI, 37(5), 349-362 (1983)), a λ operator (Erik Remaut etal, Gene, 15, 81-93 (1981)), an operator of a phosphatase operon (HideoShinagawa et al, Journal of Bacteriology, Japan, 40, 211 (1985)), andothers. The above-mentioned references are incorporated herein the sameas if each were individually reproduced in its entirety at thislocation.

The base sequence (c) is a base sequence comprising 4 base pairs rich inadenine (A) and guanine (G). In particular, the upstream 2 base pairsare rich in A and the downstream 2 base pairs are rich in G. Thedistance between the base sequence (d) and the base sequence (c) is 7base pairs or more but when the number of base pairs is large (exceeds20 base pairs), the efficiency of desired gene expression declines. Apreferred range of base pair distance is 10 to 15 base pairs betweenbase sequence (d) and base sequence (c).

The investigations by the present inventors revealed that in the basesequence (d), ATG or GTG functions as a translation initiation codon inCoryneform bacteria.

The gene to be expressed, which is directly ligated to the base sequence(d), may be any gene derived from microorganisms such as bacteriabelonging to the genus Coryneform, bacteria belonging to the genusStreptomyces, yeast belonging to the genus Saccharomyces, bacteriabelonging to the genus Escherichia and Bacillus, and so on, genesderived from animals, artificially synthesized genes, and the like.Further, the expression products of the gene to be expressed are enzymeswhich participate in production of amino acids, glucose, vitamins, etc.,improvement in quality of oils and fats, proteins, starch, etc.,modification of antibiotics, chemical substances, etc. such ashydrolase, transaminase, decarboxylase, phosphotransferase, etc., aswell as immune modulators such as lymphokines, etc., physiologicallyactive proteins such as animal hormones, etc.

As the gene coding for the repressor protein, any gene can be used aslong as the protein the gene encodes can bind to the base sequence (b)of the operator used in Coryneform bacteria and the gene has a propertysuch that the binding activity can be artificially controlled. Such agene includes, of course, a repressor gene derived from Coryneformbacteria, various repressor genes derived from Escherichia coli, e.g.,the lac repressor (lac I) (Miller and Reznikoffs, The Operon, ColdSpring Harbor Lab., pp. 31-88 (1978)), the trp repressor (trp R) (R. L.Kelley and C. Yanofsky, Proc. Natl. Acad, Sci. USA, 79, 3120-3124(1982)), the temperature-sensitive λ repressor (cI 857) (J. J. Sninskyet al., Gene, 16, 275-286 (1982)), the repressor of the phosphataseoperon (H. Shinagawa et al., J. Mol. Biol., 168, 477-488 (1983)), and soon. As means for artificially regulating the binding of these repressorproteins to the operator site, addition of chemicals, regulation ofphysical conditions such as temperature, and others may be used. Forexample, the binding of the lac repressor to the lac operator can beinhibited by addition of isopropyl-β-thiogalactopyranoside (IPTG); thebinding of the trp repressor to the trp operator can be inhibited byaddition of indole acrylic acid (IAA); the binding of thetemperature-sensitive λ repressor to the λ operator can be inhibited byincreasing culture temperature to 37° C. or higher; and the binding ofthe repressor of the phosphatase operon to the operator can be inhibitedby lowering phosphate ion concentration in the medium.

By inhibiting the binding between the repressor and the operator by theforegoing methods, the promoter inactivated by the repressor can beactivated so that the gene which is initially not expressed can bechanged to a state such that it is expressed.

The base sequence (b) functioning as the operator and the gene codingfor the repressor protein can be incorporated into the same plasmid;alternatively, they can be independently incorporated into two differentplasmids capable of co-existing in the same host and the two plasmidscan co-exist in the same host cell. In any event, it is sufficient thatthe repressor protein be capable of binding the operator in host cells.

Further, a gene the expression of which confers resistance to anantibiotic, or the like acting as a selection marker is generallyinserted in the plasmid(s).

To introduce the obtained plasmid(s) into Coryneform bacterial cells,conventional methods such as the protoplast method, among others, areapplicable.

The invention now being generally described, the same will be betterunderstood by reference to certain specific examples which are includedherein for purposes of illustration only and are not intended to belimiting of the invention or any embodiment thereof, unless specified.

EXAMPLES Example 1

Formation of pEB 001

Promoter-probing vector pEB 001 carrying a chloramphenicol-resistantgene which had lost a promoter sequence, being capable of replicating incells of Coryneform bacteria, expressing kanamycin resistance, andcarrying a site cleavable by Cla I and Hind III as a site capable ofhaving inserted therein a promoter was formed by the method shown inFIG. 1. That is, plasmid pUC 4K (cf. Gene, 19, 259-268 (1982), and J.Mol. Biol., 147, 217-226 (1981)) carrying a kanamycin-resistant genederived from transposon Tn 903 was first cleaved with Pst I to excise aDNA fragment of about 1.4 kb containing a kanamycin-resistant gene.After mixing with DNA of plasmid pCM 7 (cf. Gene, 20, 305-316 (1982)),containing a structural gene region of a chloramphenicol-resistant genecleaved with Pst I, ligation was performed using T₄ DNA ligase. Theobtained recombinant DNA was introduced into Escherichia coli HB101strain to select a strain having kanamycin resistance. From theseparated strain, plasmid DNA was obtained. It was confirmed that thekanamycin-resistant gene had been correctly inserted by the size of theplasmid and the fact that a DNA fragment of about 1.4 Kb could beobtained by cleavage with Pst I. This plasmid was named pAJ 2401A. Next,in order to impart a replication ability in cells of Coryneform bacteriato pAJ 2401A, DNA of plasmid pHM 1519 (cf. Published Unexamined JapanesePatent Application 77895/83) capable of replicating in cells ofCoryneform bacteria was cleaved with Bgl II followed by mixing with DNAof pAJ 2401A cleaved with BamH I and ligating using T₄ DNA ligase. Theobtained recombinant DNA was introduced into Escherichia coli HB101strain to select a strain having a kanamycin resistance. From theseparated strain, plasmid DNA was obtained. It was confirmed that it wasthe desired plasmid by the size of the plasmid and by lack of cleavageby BamH I and Bgl II. This plasmid was named pEB 001. pEB 001 wasintroduced into Brevibacterium lactofermentum AJ 12036 (FERM BP-734) andCorynebacterium glutamicum ATCC 13060 and a strain having a kanamycinresistance was selected. From the separated strain, plasmid DNA wasobtained. It was confirmed that it was DNA of pEB 001 by the size of theplasmid and cleavage patterns with restriction enzymes such as Pst I,BamH I, etc. From these strains, the strain Brevibacteriumlactofermentum, in which a clear band of DNA of pEB 001 had beendetected, was deposited as AJ 12177 (FERM-P7942). As above, pEB 001,which is a shuttle vector capable of replicating in Escherichia coli,Brevibacterium lactofermentum and Corynebacterium glutamicum andexpressing kanamycin resistance, carries a structural gene region of thechloramphenicol-resistant gene which has lost the promoter part, andcarries a Cla I and Hind III cleavage site as a site capable ofinserting a promoter.

DNA of pCM 7 used in this example was prepared from Escherichia coliATCC 37173. DNA of pUC 4K was purchased from Pharmacia Japan Co., Ltd.Further, DNA of pHM 1519 was prepared from Corynebacterium glutamicumATCC 13058, a strain possessing this DNA.

Formation of pEB 003

Promoter-detecting vector pEB 003 carrying a structural gene region of achloramphenicol-resistant gene which had lost a promoter sequence, beingcapable of replicating in cells of Coryneform bacteria, expressingkanamycin resistance, and carrying a site cleavable with 5 restrictionenzymes (Pst I, Hpa I, BamH I, Cla I and Hind III) as a site capable ofhaving a promoter inserted therein was formed by the method shown inFIG. 2. First, DNA from pAJ 2401A was partly cleaved with Pst I. The DNAfragments formed by cleavage at one site were collected by agarose geland a single strand portion projecting at both edges of the DNAfragments was removed to produce blunt ends. Then, ligation wasperformed using T₄ DNA ligase. The obtained recombinant DNA wasintroduced into Escherichia coli HB101 strain and a strain having akanamycin resistance was selected. From the separated strain, plasmidDNA was obtained, and plasmid DNA cleaved with Pst I at one site wasselected. In these plasmids, a plasmid which had lost the Pst I cleavagesite at the 5'-side and a plasmid which had the Pst I cleavage site atthe 3'-side of the kanamycin-resistant gene were present. The formerplasmid was named pEB 002A and the latter was named DEB 002. Next, DNAof pEB 001 was cleaved with Cla I and Sal I to obtain DNA fragment Acontaining the structural gene region of the chloramphenicol-resistantgene and a replication initiation site in cells of Coryneform bacteria.On the other hand, pEB 002A was cleaved with Pst I and Sal I to obtainDNA fragment B containing a kanamycin-resistance gene. Further, anoligonucleotide as shown in FIG. 3 was synthesized according to thephosphite method to prepare DNA fragment C carrying a Pst I cleavagesite at the 5'-terminal, a Cla I cleavage site at the 3'-terminal and aHpa I and BamH I cleavage site therebetween. After mixing DNA fragmentsA, B and C, they were ligated using T₄ DNA ligase. The obtainedrecombinant DNA was introduced into Brevibacterium lactofermentum AJ12036 (FERM BP-734) and strains having a kanamycin resistance wereselected. From the separated strains, plasmid DNA was obtained. It wasconfirmed that it was the desired plasmid DNA by the size of the plasmidand the cleavage patterns with restriction enzymes such as Pst I, Hpa I,BamH I, etc. This plasmid was named pEB 003. pEB 003 possesses cleavagesites with 5 restriction enzymes (Pst I, Hpa I, BamH I, Cla I and HindIII) as sites where a promoter may be inserted. Among them, the cleavagesite with Pst I, Hpa I and BamH I is the only one in the vector which isextremely useful for insertion of a promoter. Brevibacteriumlactofermentum AJ 12036 (FERM BP-734) to which pEB 003 had beenintroduced was deposited as AJ 12178 (FERMP 7943).

Incorporation of a tac promoter and a lac operator into pEB 003

DNA of plasmid pDR 540 (cf. Gene, 20, 231 (1982)) (commerciallyavailable, purchased from Pharmacia Japan Co., Ltd.) carrying theEscherichia coli tac promoter and lac operator was cleaved with BamH Iand Pst I. The obtained DNA fragments of about 1100 base pairscontaining the tac promoter and the lac operator were fractionated andpurified by agarose gel electrophoresis. After mixing with DNA of pEB003 cleaved with Pst I and BamH I, ligation was performed using T₄ DNAligase. The obtained recombinant DNA was introduced into Escherichiacoli HB 101 strain and strains having a chloramphenicol resistance wereselected. From the separated strains, plasmid DNA was obtained. It wasconfirmed that it was a plasmid having incorporated therein theEscherichia coli tac promoter as desired, by the size of the plasmid andcleavage patterns with restriction enzymes such as Pst I, BamH I, etc.This plasmid was named pEB 003TA.

Next, pEB 003TA was introduced into Brevibacterium lactofermentum AJ12036 (FERM BP-734) and strains having a kanamycin resistance wereselected. From the separated strains, a plasmid was obtained. It wasconfirmed that the plasmid was pEB 003TA by the size of the plasmid andcleavage patterns with restriction enzymes such as Pst I, BamH I, etc.

Formation of a gene expression-regulating vector pEC 701

Plasmid pEC 701, capable of regulating expression of a gene ligateddownstream from a tac promoter and a lac operator region by the actionof a regulating gene (lac repressor) was formed by the method as shownin FIG. 4.

First, after pEB 003TA (formed as above) was cleaved with Pst I and SalI, DNA fragment D of about 5.6 kb was recovered from agarose gel. On theother hand, the plasmid pIN III (y. Masui et al., ExpermentalManipulation of Gene Expression, Ed. M. Inouye, pp. 15, Academic Press,Inc. 1983) of Escherichia coli was cleaved with Pst I, and Sal I, andDNA fragment E of about 5.6 kb containing the lac repressor (lac I) wasrecovered from agarose gel. Further, plasmid pUC 4K (Gene, 19, 256-268(1982)) of Escherichia coli was cleaved with Pst I, and DNA fragment Fof about 1.2 kb containing a kanamycin-resistant gene was likewiserecovered from agarose gel. The thus obtained 3 DNA fragments D, E and Fwere mixed in an approximately equal amount. After ligating with T₄ DNAligase, they were introduced into Brevibacterium lactofermentum AJ 12036(FERM BP-734) and strains showing a kanamycin resistance were selected.From the separated strains, plasmid DNA of about 12.4 kb composed byligation of 3 DNA fragments D, E and F was obtained. It was confirmedthat it was the desired plasmid by cleavage patterns with restrictionenzymes such as Pst I, Sal I, etc. This plasmid was named pEC 701. pEC701 was again introduced into Brevibacterium lactofermentum AJ 12036(FERM BP-734) and Corynebacterium glutamicum ATCC 13060, from whichstrains those showing a kanamycin resistance were selected. From theseparated strains, plasmid DNA was obtained. It was confirmed that allDNA was that of pEC 701 by the size of the plasmid and cleavage patternswith restriction enzymes such as Pst I, Sal I, etc.

The thus formed pEC 701 is a shuttle vector carrying a tac promoter, alac operator and a lactose operon repressor (lac repressor), beingcapable of replicating in Escherichia coli, Brevibacteriumlactofermentum and Corynebacterium glutamicum and expressing a kanamycinresistance. It is also a vector bearing a structural gene region of achloramphenicol-resistant gene, and is capable of regulating expressionof the chloramphenicol-resistant gene by the lac repressor. Further,there are sites which can be cleaved with restriction enzymes such asBamH I, etc. downstream from the lac operator. pEC 701 is also a vectorcapable of regulating expression of the gene by incorporating astructural gene region free from a promoter of any useful gene,utilizing these sites as cloning sites.

Example 2

Incorporation of a lac promoter and operator into pEB 003

DNA of plasmid pGL 101 (cf. J. Mol. Appl. Genet., 1, 139 (1981))carrying the Escherichia coli lac UV5 promoter and operator was cleavedwith Pst I and Pvu II. The obtained DNA fragments of about 1000 basepairs containing the lac UV5 promoter and operator were fractionated andpurified by agarose gel electrophoresis. After mixing with the fragmentscontaining the lac UV promoter and operator and DNA of pEB 003 cleavedwith Pst I and Hpa I, ligation was performed using T₄ DNA ligase. Theobtained recombinant DNA was introduced into Escherichia coli HB 101strain and strains having a chloramphenicol resistance were selected.From the separated strains, plasmid DNA was obtained. It was confirmedthat it was a plasmid having incorporated therein the Escherichia colilac UV5 promoter and operator as desired, by the size of the plasmid andcleavage patterns with restriction enzymes such as Pst I, BamH I, etc.This plasmid was named pEB 003LA.

Next, pEB 003LA was introduced into Brevibacterium lactofermentum AJ12036 (FERM BP-734) and strains having a kanamycin resistance wereselected. From the separated strains, a plasmid was obtained. It wasconfirmed that the plasmid was pEB 003LA by the size of the plasmid andcleavage patterns with restriction enzymes such as Pst I, BamH I, etc.

Formation of a gene expression-regulating vector pEC 702

Plasmid pEC 702, capable of regulating expression of a gene ligateddownstream from a lac promoter and lac operator region by the action ofthe lac repressor, was formed by the method shown in FIG. 5. After pEB003LA was cleaved with Pst I and Sal I, DNA fragment D' of about 5.5 kbcontaining the lac promoter and operator was recovered from agarose gel.After ligating the fragment D', the Pst I-Sal I fragment E of about 5.6kb containing the lac repressor (lac I) excised from plasmid pIN II andthe Pst I fragment F of about 1.2 kb containing a kanamycin-resistantgene excised from pUC 4K using T₄ DNA ligase in a manner similar toExample 1, they were introduced into Brevibacterium lactofermentum AJ12036 (FERM BP-734) and strains showing a kanamycin resistance wereselected. From the separated strains, plasmid DNA of about 12.3 kb,composed of 3 ligated DNA fragments D', E and F, was obtained. It wasconfirmed that it was the desired plasmid by cleavage patterns withrestriction enzymes such as Pst I I, Sal I, etc. This plasmid was namedpEC 702. pEC 702 was again introduced into Brevibacterium lactofermentumAJ 12036 (FERM BP-734) and Corynebacterium glutamicum ATCC 13060, fromwhich strains those showing a kanamycin resistance were selected. Fromthe separated strains, plasmid DNA was obtained. It was confirmed thatall of this DNA was that of pEC 702 by the size of the plasmid andcleavage patterns with restriction enzymes such as Pst I, Sal I, etc.

The thus formed pEC 702 is a shuttle vector carrying a lac promoter, alac operator and a lac repressor, being capable of replicating inEscherichia coli, Brevibacterium lactofermentum and Coryneform, andexpressing a kanamycin resistance. It is also a vector bearing astructural gene region of a chloramphenicol-resistant gene and iscapable of regulating expression of the chloramphenicol-resistant geneby the lac repressor. Further, there are sites which may be cleaved byrestriction enzymes such as BamH I, etc. downstream from the lacoperator. pEC 701 is also a vector capable of regulating expression ofthe gene by incorporating a structural gene region of any useful genefree from a promoter utilizing these sites as cloning sites.

Example 3

Regulation of gene expression using the gene expression-regulatingvectors pEC 701 and pEC 702

Brevibacterium lactofermentum AJ 12036 (FERM-BP 734) harboring pEC 701or pEC 702, was inoculated on 50 ml of liquid medium of pH 7.2containing 1% yeast extract, 1% polypeptone, 0.5% NaCl, 0.5% glucose and10 μg/ml of kanamycin, followed by shake culture at 30° C. Afterculturing for 3 hours, 0.2 mM of isopropyl-β-thiogalactopyranoside(IPTG) was supplemented thereto and culture was continued. With passageof time, 5 ml samples of the culture solution were taken and bacteriawere collected by centrifugation. After washing, the cells were groundby way of ultrasonic waves and centrifuged at 15,000 r.p.m. for 15minutes. The supernatant was used as a crude enzyme solution andmeasured with respect to chloramphenicol acetyltransferase activity. Thechloramphenicol acetyltransferase activity was measured by the method ofW. V. Shaw, Methods in Enzymology, 43, 737 (1975). Similar runs werealso performed on Brevibacterium lactofermentum AJ 12036 having noplasmid, AJ 12036 strain harboring plasmid pEB 003TA having a tacpromoter and a lac operator but having no lac repressor, and AJ 12036strain harboring plasmid pEB 003LA having a lac promoter and a lacoperator but having no lac repressor. The results are shown in Table 1.It was verified that by the addition of IPTG, the chloramphenicolacetyltransferase activity was enhanced by about 10 fold with the pEC701-harboring strain and the pEC 702-harboring strain, respectively, ascompared to the case where none is added, and gene expression ofchloramphenicol acetyltransferase could be artificially regulated by theaddition of IPTG in both the pEC 701-harboring strain and the pEC702-harboring strain. On the other hand, in both the pEB 003TA-harboringstrain and the pEB 003LA-harboring strain, high activity was notedirrespective of the presence or absence of IPTG and regulation of geneexpression was impossible.

                  TABLE 1                                                         ______________________________________                                        Regulation of chloramphenicol acetyltransferase gene in                       Brevibacterium lactofermentum AJ 12036 using the gene-                        expression regulating plasmids pEC 701 and pEC 702                                          Activity of Chloramphenicol                                                   Acetyltransferase                                               Plasmid     IPTG    0 hr       1 hr 2 hr                                      ______________________________________                                        None        -        0          0    0                                                    +        0          0    0                                        pEB 003TA   -       470        460  320                                                   +       470        460  300                                       pEC 701     -        18         23   28                                                   +        18        101  205                                       pEB 003LA   -       168        166  160                                                   +       168        165  155                                       pEC 702     -        6          6    5                                                    +        6          42   36                                       ______________________________________                                    

Example 4

Incorporation of a trp promoter and a trp operator into pEB 003

DNA of plasmid pDR 720 (cf. Gene, 20 231 (1982)) (commerciallyavailable, purchased from Pharmacia Japan Co., Ltd.) carrying theEscherichia coli trp promoter and trp operator was cleaved with Pst I.The obtained DNA fragments of about 1100 base pairs containing the trppromoter and trp operator were fractionated and purified by agarose gelelectrophoresis. After mixing the DNA fragments with DNA of pEB 003cleaved with Pst I, ligation was performed using T₄ DNA ligase. Theobtained recombinant DNA was introduced into Escherichia coli HB 101strain and strains having a chloramphenicol resistance were selected.From the separated strains, plasmid DNA was obtained. It was confirmedthat it was a plasmid having incorporated therein the Escherichia colitrp promoter and trp operator as desired, by the size of the plasmid andcleavage patterns with restriction enzymes such as Pst I, Pvu I, etc.This plasmid was named pEB 003TR.

Next, pEB 003TR was introduced into Brevibacterium lactofermentum AJ12036 (FERM BP-734) and strains having a kanamycin resistance wereselected. From the separated strains, a plasmid was obtained. It wasconfirmed that the plasmid was pEB 003TR by the size of the plasmid andcleavage patterns with restriction enzymes such as Pst I, Pvu I, etc.

Formation of a gene expression-regulating vector pEC 801

Plasmid pEC 801 capable of regulating expression of a gene ligateddownstream from a trp promoter and trp operator region by the action ofa regulating gene (trp repressor) was formed by the method shown in FIG.6. First, pEB 003TR (formed as above) was cleaved with Sal I. On theother hand, Escherichia coli plasmid ptrp R3 (W. Roeder and R. L.Somerville, Mol. Gene. Genet., 176, 361 (1979)) was cleaved with BamH Iand, about 1200 base pairs of DNA fragments containing the trp repressor(trpR) were recovered from agarose gel. The thus obtained two DNA's wereligated with T₄ DNA ligase via a synthesized DNA adaptor synthesized bythe phosphite method. Thereafter, they were introduced intoBrevibacterium lactofermentum AJ 12036 (FERM BP-734) and strains showinga kanamycin resistance were selected. From the separated strains,plasmid DNA of about 9.7 kb composed by ligation of the above-described2 DNA fragments was obtained. It was confirmed that it was the desiredplasmid by the cleavage pattern with BamH I. This plasmid was named pEC801. pEC 801 was again introduced into Brevibacterium lactofermentum AJ12036 (FERM BP-734) and Corynebacterium glutamicum ATCC 13060, fromwhich strains those showing a kanamycin resistance were selected. Fromthe separated strains, plasmid DNA's were obtained. It was confirmedthat all of these were DNA of pEC 801 by the size of the plasmid and thecleavage pattern with BamH I.

The thus formed pEC 801 is a shuttle vector carrying a trp promoter, atrp operator and a trp repressor, which is capable of replicating inEscherichia coli, Brevibacterium lactofermentum and Corynebacteriumglutamicum and expressing a kanamycin resistance. It is also a vectorbearing a structural gene region of a chloramphenicol-resistant gene andis capable of regulating expression of the chloramphenicol-resistantgene by the trp repressor. Further, there are sites which may be cleavedwith restriction enzymes such as Pst I, Hpa I, BamH I, etc. downstreamfrom the trp promoter. pEC 701 is also a vector capable of regulatingexpression of the gene by incorporating a structural gene region freefrom a promoter, of any useful gene, utilizing these sites as cloningsites.

Regulation of gene expression using the gene express-regulating vectorpEC 801

Brevibacterium lactofermentum AJ 12036 (FERM-BP 734) harboring pEC 801was inoculated on 50 ml of liquid medium of pH 7.2 containing 1% yeastextract, 1% polypeptone, 0.5% NaCl, 0.5% glucose and 10 μg/ml ofkanamycin followed by shake culture at 30° C. After culturing for 3hours, 0.13 mM of indole acrylic acid (IAA) was supplemented and culturewas continued. With passage of time, 5 ml samples of the culturesolution were taken and bacteria were collected by centrifugation. Afterwashing, the cells were ground by way of ultrasonic waves andcentrifuged at 15,000 r.p.m. for 15 minutes The supernatant was used asa crude enzyme solution and measured with respect to chloramphenicolacetyltransferase activity. The chloramphenicol acetyltransferaseactivity was measured by the method of W. V. Shaw, Methods inEnzymology, 43, 737 (1975). Similar runs were also performed onBrevibacterium lactofermentum AJ 12036 having no plasmid, the AJ 12036strain harboring plasmid pEB 003TR having a trp promoter and a trpoperator but having no trp repressor. The results are shown in Table 2.It was verified that by the addition of IAA, the chloramphenicolacetyltransferase activity was enhanced by about 5 fold with the pEC801-harboring strain, as compared to the case where none is added, andgene expression of chloramphenicol acetyltransferase could beartificially regulated by the addition of IAA in the pEC 801-harboringstrain. On the other hand, in the case of a pEB 003TR-harboring strainhaving no trp repressor, high activity was noted irrespective of thepresence or absence of IAA and regulation of gene expression wasimpossible.

                  TABLE 2                                                         ______________________________________                                        Regulation of chloramphenicol acetyltransferase gene in                       Brevibacterium lactofermentum AJ 12036 using the gene-                        expression regulating plasmid pEC 801                                                       Activity of Chloramphenicol                                                   Acetyltransferase                                               Plasmid     IAA     0 hr       1 hr 2 hr                                      ______________________________________                                        None        -        0          0    0                                                    +        0          0    0                                        pEB 003TR   -       252        256  251                                                   +       252        255  250                                       pEC 801     -        15         21   23                                                   +        15        100  102                                       ______________________________________                                         (unit: nmol/min. mg)                                                     

Example 5

Formation of a gene expression-regulating vector pEC 901

Plasmid pEC 901 capable of regulating expression of a gene ligateddownstream from a P_(R) P_(L) promoter and operator region of lambdaphage by the action of a temperature-sensitive cI 857 repressor wasformed by the method as shown in FIG. 7. After pMY 12-6 (Mol. Gen.Genet., 187, 79-86 (1982)) was cleaved with Pst I and BamH I, DNAfragments of about 1.35 kb containing a P_(R) P_(L) promoter operatorand cI 857 repressor were recovered from agarose gel. On the other hand,pEP 003 was cleaved with Pst I and BamH I, and DNA fragments of about7.4 kb were recovered from agarose gel. The two DNA fragments wereligated with T₄ DNA ligase and then introduced into Escherichia coli HB101, and strains showing a kanamycin resistance were selected. From theseparated strains, plasmid DNA of about 8.7 kb formed by ligation of the2 DNA fragments was obtained. It was confirmed that it was the desiredplasmid by cleavage patterns with restriction enzymes such as Pst I,BamH I, etc. This plasmid was named pEC 901. pEC 901 was introduced intoBrevibacterium lactofermentum AJ 12036 (FERM BP-734) and Corynebacteriumglutamicum ATCC 13060, from which strains those showing a kanamycinresistance were selected. From the separated strains, plasmid DNA wasobtained. It was confirmed that all of the DNA was that of pEC 901 bythe size of the plasmid and cleavage pattern with restriction enzymessuch as Pst I, BamH I, etc.

The thus formed pEC 901 is a shuttle vector carrying a lambda phageP_(R) P_(L) promoter, operator and cI 857 repressor, being capable ofreplicating in Escherichia coli, Brevibacterium lactofermentum andCorynebacterium glutamicum and expressing a kanamycin resistance. It isalso a vector bearing a structural gene region of achloramphenicol-resistant gene and capable of regulating expression ofthe chloramphenicol-resistant gene by temperature using the cI 857repressor. Further, there are sites which may be cleaved withrestriction enzymes such as BamH I, Cla I, etc. downstream from theP_(R) P_(L) promoter. pEC 901 is also a vector capable of regulatingexpression of the gene by incorporating a structural gene region of anyuseful gene free from a promoter, utilizing these sites as cloningsites.

Regulation of gene expression using the gene expression-regulatingvector pEC 901

Brevibacterium lactofermentum AJ 12036 (FERM-BP 734) harboring pEC 901was inoculated on 50 ml of liquid medium of pH 7.2 containing 1% yeastextract, 1% polypeptone, 0.5% NaCl, 0.5% glucose and 10 μg/ml ofkanamycin followed by shake culture at 30° C. When the OD₅₇₀ reached0.4, the culture solution was treated at 43° C. for 5 minutes followedby shake culture at 39° C. With passage of time, 20 ml samples of theculture solution were taken and bacterial cells were collected bycentrifugation. After washing, the cells were ground via ultrasonicwaves and centrifuged at 15,000 r.p.m. for 15 minutes. The supernatantwas used as a crude enzyme solution and measured with respect tochloramphenicol acetyltransferase activity. The chloramphenicolacetyltransferase activity was measured by a method similar to Example3. Similar runs were also performed on Brevibacterium lactofermentum AJ12036 having pEB 003. The results are shown in Table 3. By elevating thetemperature, the chloramphenicol acetyltransferase activity wasexpressed in the pEC 901-harboring strain and in the case where notemperature was elevated, the activity was not observed. On the otherhand, no enhancement of the chloramphenicol acetyltransferase activitydue to elevation of the temperature was noted in the case of the pEB003-harboring strain.

                  TABLE 3                                                         ______________________________________                                        Regulation of chloramphenicol acetyltransferase gene in                       Brevibacterium lactofermentum AJ 12036 using the gene-                        expression regulating plasmid pEC 901                                                         Activity of Chloramphenicol                                          Culture  Acetyltransferase                                             Plasmid  Temperature                                                                              0 hr       6 hr 24 hr                                     ______________________________________                                        pEB 003  30° C.                                                                            29         33   18                                                 40° C.                                                                            29         17   17                                        pEC 901  30° C.                                                                             0          0    0                                                 40° C.                                                                             0         80   103                                       ______________________________________                                         (unit: nmol/min. mg)                                                     

Example 6

Regulation of gene expression in the presence of more than one plasmid

With respect to a method for regulating gene expression by incorporatingan operator and a gene coding for a repressor protein in separateplasmids and having them co-exist in the same host cell, an exampleusing a trp operator and a trp repressor is shown below. However, thecombination of operator and repressor is not limited to the combinationof a trp operator and a trp repressor shown in this example but may beany combination of a lac operator and a lac repressor, lambda phage,P_(R) P_(L) operator and lambda phage repressor (cI 857 repressor,etc.), or the like.

As a plasmid bearing a trp promoter, trp operator and chloramphenicolacetyltransferase gene, pEB 003TR formed in Example 4 was used. In thecase where pEB 003TR alone was introduced into Brevibacteriumlactofermentum AJ 12036 (FERM BP-734), it was impossible to regulate thechloramphenicol acetyltransferase activity, as shown in Example 4.

As the plasmid bearing a trp repressor gene, any repressor can be usedas long as it can co-exist with pEB 003TR in Coryneform bacterial cellsand produces a trp repressor. For example, pEC 830 obtained byincorporating a trp repressor gene into trimethoprim-resistant plasmidvector pAJ 228, and the like, can be used.

Formation of plasmid vector pAJ 228

(1) Trimethoprim-resistant variant AJ 12146 (FERM-P 7672) derived frommutation of Brevibacterium lactofermentum AJ 12036 was inoculated on a 1liter CM2G medium (1 g/dl of peptone, 1 g/dl of yeast extract, 0.5 g/dlof glucose and 0.5 g/dl of NaCl, adjusted pH to 7.2) followed by shakeculture at 30° C. for about 3 hours. Bacterial cells were collected atthe exponential growth phase. After the cells were lysed withlysozyme-SDS, chromosomal DNA was extracted and purified by conventionaltreatment with phenol to finally obtain 3.0 mg of DNA.

Brevibacterium lactofermentum AJ 12146 was obtained by contactingBrevibacterium lactofermentum AJ 12036 with 1,000 μg/ml ofN-methyl-N'-nitro-N-nitrosoguanidine at 0° C. for 20 minutes forvariation treatment and separating a strain capable of growing inminimum medium (containing 2 g/dl of glucose, 1 g/dl of ammoniumsulfate, 0.25 g/dl of urea, 0.1 g/dl of KH₂ PO₄, 0.04 g/dl of MgSO₄. 7H₂O, 200 μg/l of thiamine hydrochloride, 50 μg/l of biotin, 2 ppm of ironirons, 2 ppm of manganese ions and 1.5 g/dl of agar, pH adjusted to 7.0)supplemented with 100 μg/ml of trimethoprim.

(2) As the vector, pAM 330 was used. pAM 330 was prepared as follows:

First, Brevibacterium lactofermentum ATCC 13869 having pAM 330 asplasmids was inoculated on 100 ml of CM2G medium. After culturing at 30°C. to reach late exponential growth phase, the cells were lysed bylysozyme and SDS. The supernatant was obtained by ultracentrifugation of30,000× g for 30 minutes. After treatment with phenol, 2 volumes ofethanol were added to recover DNA as a precipitate. After the DNA wasdissolved in a small quantity of TEN buffer (20 mM tris-hydrochloride,20 mM NaCl, 1 mM EDTA, pH 8.0), the solution was subjected to agarosegel electrophoresis for separation. Then, the separated product wastaken out to obtain about 15 μg of pAM 330 DNA.

(3) The chromosomal DNA, 20 μg, obtained in (1) and 10 μg of the plasmidDNA obtained in (2) were treated with restriction endonuclease Mbo I at37° C. for 30 minutes, respectively, to effect partial cleavage. Afterheat treatment at 65° C. for 10 minutes, both reaction solutions weremixed with each other, and the mixture was subjected to a ligationreaction of DNA strands with T₄ phage-derived DNA ligase at 10° C. for24 hours in the presence of ATP and dithiothreitol. After heat treatmentat 65° C. for 10 minutes, a 2-fold volume of ethanol was added to thereaction solution to precipitate and harvest DNA after completion of theligation reaction.

(4) Brevibacterium lactofermentum AJ 12036 sensitive to trimethoprim wasused as recipient. As the transformation method, a protoplasttransformation method was used. First, the cells were cultured in 5 mlof CM2G medium to reach an early exponential growth phase. After adding0.6 units/ml of penicillin, shake culture was conducted for anadditional 1.5 hours. Cells were harvested by centrifugation and washedwith 0.5 ml of SMMP medium (pH 6.5) composed of 0.5M sucrose, 20 mMmaleic acid, 20 mM magnesium chloride and 3.5% Pennassay broth (Difco)and then suspended in SMMP medium containing 10 mg/ml of lysozyme. Thesuspension was treated at 30° C. for 20 hours to obtain protoplasts.After centrifuging at 6000× g for 10 minutes, the protoplasts werewashed with SMMP and resuspended in 0.5 ml of SMMP. The thus obtainedprotoplasts were mixed with 10 μg of DNA prepared in (3) in the presenceof 5 mM EDTA. After polyethylene glycol was added to the mixture toreach the final concentration of 30%, the mixture was allowed to standat room temperature for 2 minutes to incorporate DNA into theprotoplasts. After the protoplasts were washed with 1 ml of SMMP medium,they were resuspended in 1 ml of SMMP, and the suspension was culturedat 30° C. for 2 hours for phenotypic expression. The culture solutionwas spread over a protoplast renaturation medium of pH 7.0. Therenaturation medium contained, per one liter of distilled water, 12 g oftris(hydroxymethyl)aminomethane, 0.5 g of KCl, 10 g of glucose, 8.1 g ofMgCl₂.6H₂ O, 2.2 g of CaCl₂.2H₂ O, 4 g of peptone, 4 g of yeast extractpowder, 1 g of Casamino Acid (Difco), 0.2 g of K₂ HPO₄, 135 g of sodiumsuccinate, 8 g of agar and 25 μg/ml of trimethoprim (Sigma Co., Ltd.).

After culturing at 30° C. for 1 week, about 100 colonies appeared, whichwere transferred to minimum medium plates which contained 2% glucose, 1%ammonium sulfate, 0.25% urea, 0.1% KH₂ PO₂, 0.04% MgSO₄.7H₂ O, 2 ppm ofiron ions, 2 ppm of manganese ions, 200 μg/l thiamine hydrochloride and50 μg/l biotin, pH 7.0, 1.8% agar and 50 μg/ml trimethoprim) by thereplica plating method to obtain a strain resistant to trimethoprim.

(5) From the strain, a lysate was predated by the method described in(2). When plasmid DNA was detected by agarose gel electrophoresis, aplasmid obviously larger than vector pAM 330 was detected. This strainwas named AJ 12147 (FERM-P 7673).

(6) To confirm that the trimethoprim-resistant genes were present onplasmids (pAJ 228) possessed by AJ 12147, Brevibacterium lactofermentumAJ 12036 was again transformed using the plasmid DNA.

Among colonies which appeared and which had trimethoprim-resistance, 10colonies each were chosen and collected and the plasmid DNA was detectedby agarose gel electrophoresis. In all of them, plasmids having the samesize as that of pAJ 228 were present. It became clear that genesexpressing trimethoprim resistance were present on the recombinantplasmids described above.

(7) Properties of pAJ 228 DNA

(a) The molecular weight of pAJ 228 was determined by agarose gelelectrophoresis. Agarose gel electrophoresis was performed in accordancewith the method of P. A. Sharp, et al. (Biochemistry, 12, 3055 (1973))using 0.8% gel at a constant voltage of 5 V per cm of gel length for 15hours. The molecular weight was calculated by comparing mobility withthat of a molecular weight marker having a known molecular weight: λphage Hind III fragment (BRL Co., Ltd.) subjected to reaction with 0.5units of restriction enzyme Cla I for cleaving 1 portion of pAJ 228 with0.5 μg of pAJ 228 at 37° C. for 1 hour, which was determined to be 7.6kb.

(b) Preparation of a restriction map of pAJ 228 DNA

A commercially available restriction enzyme from BRL was used. Cleavageof pAJ 228 DNA with the restriction enzyme was carried out using atleast a 3 fold excess of enzyme under given conditions with respect toeach enzyme. In the case where plasmid DNA was cleaved with one or morerestriction enzymes for the purpose of a restriction map, fragmentscleaved with a first restriction enzyme were isolated by agarose gel forseparation in accordance with the method of Tanaka et al. (T. Tanaka andB. Weisblum, J. Bacteriol., 121, 354 (1975)), condensed by ethanolprecipitation and then cleaved with a second restriction enzyme. Thecleaved fragments were subjected to agarose gel electrophoresis andtheir molecular weights were calculated to prepare a restriction map(FIG. 8).

Formation of a gene expression-regulating vector pEC 830

A trimethoprim-resistant vector pEC 830 for regulating gene expressionhaving incorporated therein a trp repressor, was formed by the methodshown in FIG. 9. First, plasmid ptrp R3 was cleaved with BamH I as inExample 4, and about 1200 base pairs of DNA fragments containing a trprepressor (trp R) were recovered from agarose gel. Next, the DNAfragments were reacted with DNA polymerase (Klenow fragment) in thepresence of 4 deoxynucleotides (dATP, dGTP, dCTP, dTTP) to convert theedges of the fragments into smooth terminals. The thus prepared DNAfragments and DNA of pAJ 228 cleaved with Hpa I were mixed. Afterligating them using T₄ DNA ligase, they were introduced intoBrevibacterium lactofermentum AJ 12036 (FERM BP-734), and strains havinga trimethoprim resistance were selected. Plasmids present in theseparated strains were extracted. It was confirmed that it was thedesired plasmid of about 8.8 kb by the size. This plasmid was named pEC830. pEC 830 was again introduced into Brevibacterium lactofermentum AJ12036 (FERM BP-734) and strains having a trimethoprim resistance wereselected. From the separated strains, plasmid DNA was obtained. It wasconfirmed that both were DNA of pEC 830 by the size of the plasmid andcleavage pattern with Xba I.

Regulation of gene expression using the gene expression-regulatingvector pEC 830

pEC 830 DNA was introduced into Brevibacterium lactofermentum AJ 12036(FERM-BP 734) harboring plasmid pEB 003TR carrying a trp promoter and atrp operator formed in Example 4. The thus obtained strain harboringboth pEB 003TR and pEC 830 was inoculated on 50 ml of liquid mediumcontaining 10 μg/ml of kanamycin and 50 μg/ml of trimethoprim and havinga composition of 1% yeast extract, 1% polypeptone, 0.5% NaCl and 0.5%glucose followed by shake culture at 30° C. After culturing for 3 hours,0.13 mM of indole acrylic acid (IAA) was supplemented and culture wascontinued. With passage of time, 5 ml samples of the culture solutionwere taken and the chloramphenicol acetyltransferase activity wasmeasured by the method shown in Example 4. Similar runs were alsoperformed on the AJ 12036 strain carrying pEB 003TR or pEC 830 singlyand the AJ 12036 strain harboring no plasmid. The results are shown inTable 4. It was verified that by the addition of IAA, thechloramphenicol acetyltransferase activity was enhanced by about 3 to 4fold with the strain harboring both pEB 003TR and pEC 830, as comparedwith the case of no addition of IAA. Thus, the gene expression could beartificially regulated by the addition of IAA. On the other hand, in thecase of the strain harboring pEB 003TR singly, high activity was notedirrespective of the presence or absence of IAA, and regulation of geneexpression was impossible.

                  TABLE 4                                                         ______________________________________                                        Regulation of chloramphenicol acetyltransferase gene in                       Brevibacterium lactofermentum AJ 12036 using the gene-                        expression regulating plasmid pEC 830                                                       Activity of Chloramphenicol                                                   Acetyltransferase                                               Plasmid     IAA     0 hr       1 hr 2 hr                                      ______________________________________                                        None        -        0          0    0                                                    +        0          0    0                                        pEB 003TR   -       210        225  218                                                   +       200        230  230                                       pEC 830     -        0          0    0                                                    +        0          0    0                                        pEB 003TR,  -        25         27   28                                       pEC 830     +        25         95  110                                       ______________________________________                                         (unit: nmol/min. mg)                                                     

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A recombinant plasmid vector which replicatesand expresses in a coryneform bacterial cell, comprising:a) a basesequence obtained from E. coli which functions as a promoter in acoryneform bacterium; b) a base sequence obtained from E. coli whichfunctions as an operator downstream from said base sequence a), saidoperator being selected from the group consisting of a base sequence ofa lac operator, a trp operator, a λ operator and an operator of aphosphatase operon; c) a base sequence obtained from E. coli whichfunctions as a site for ribosome binding in a coryneform bacterium; andd) a base sequence functioning as a translation initiation codon and aheterologous gene which is operably linked to said base sequence a),said vector further comprising a gene obtained from E. coli coding for arepressor protein which binds to said sequence b) functioning as anoperator, and wherein the binding of said repressor protein isartificially regulated in a growth medium.
 2. A bacterium containingtherein the plasmid vector of claim 1, comprising:a) a base sequencefunctioning as a promoter in a coryneform bacterium; b) a base sequencefunctioning as an operator downstream from said base sequence a), saidoperator being selected from the group consisting of a base sequence ofa lac operator of a trp operator, a λ operator and an operator of aphosphatase operon; c) a base sequence functioning as a site forribosome binding in a coryneform bacterium; and d) a base sequencefunctioning as a translation initiation codon and a heterologous gene,which is operably linked to said base sequence a), said vector furthercomprising a gene coding for a repressor protein which binds to saidsequence b) functioning as an operator, and wherein the binding of saidrepressor protein is artificially regulated in a growth medium.
 3. Thevector of claim 1, wherein said translation initiation codon is ATG orGTG.
 4. The vector of claim 1, wherein said heterologous gene isobtained from Streptomyces, Saccharomyces, Escherichia, or Bacillus. 5.The vector of claim 1, wherein said gene obtained from E. coli codingfor a repressor protein is obtained from lac repressor, trp repressor,temperature sensitive λ repressor, or the repressor of phosphataseoperon.
 6. A recombinant plasmid vector which replicates and expressesin a coryneform bacterial cell, comprising:a) a base sequence obtainedfrom E. coli which functions as a promoter in a coryneform bacterium; b)a base sequence obtained from E. coli which functions as an operatordownstream from said base sequence a), said operator being selected fromthe group consisting of a base sequence of a lac operator, a trpoperator, a λ operator and an operator of a phosphatase operon; c) abase sequence obtained from E. coli which functions as a site forribosome binding in a coryneform bacterium; and d) a base sequencefunctioning as a translation initiation codon and a heterologous gene,which is operably linked to said base sequence a).
 7. A bacteriumcontaining therein the plasmid vector of claim 6, which replicates andexpresses in a coryneform bacterial cell, comprising:a) a base sequenceobtained from E. coli which functions as a promoter in a coryneformbacterium; b) a base sequence obtained from E. coli which functions asan operator downstream from said base sequence a), said operator beingselected from the group consisting of a base sequence of a lac operator,a trp operator, a λ operator and an operator of a phosphatase operon; c)a base sequence obtained from E. coli which functions as a site forribosome binding in a coryneform bacterium; and d) a base sequencefunctioning as a translation initiation codon and a heterologous gene,which is operably linked to said base sequence a), wherein saidbacterium contains a gene obtained from E. coli coding for a repressorprotein which binds to said sequence b) functioning as an operator, andwherein the binding of said repressor protein is artificially regulatedin a growth medium.
 8. The vector of claim 7, wherein said translationinitiation codon is ATG or GTG.
 9. The vector of claim 7, wherein saidheterologous gene is obtained from Streptomyces, Saccharomyces,Escherichia, or Bacillus.